DESCRIPTION (Applicant's abstract): In recent years, oxidative stress has been shown to have critical roles in the pathogenesis of vascular disease Reactive oxygen species (ROS) such as superoxide (02-) and hydrogen peroxide (H202) have profound effect on vascular smooth muscle cell (VSMC) growth and migration, endothelial function and inflammation. In the previous grant period, we focused on the NAD(P)H oxidase, an enzyme that is the major source of 02- vascular cells. We showed that this enzyme is p22phox-based, but structurally unique from the neutrophil NAD(P)H oxidase, and that it is absolutely required for VSMC growth and hypertrophy. In addition, we cloned a new oxidase (subunit) from VSMC, termed nox- 1, the first of what is now a family of gp9 1 phox homologues expressed in non-phagocytic cells. We now propose to further define the subunit structure of this oxidase and to gain insight into its growth-related downstream molecular targets. Inthe first specific aim, we plan to determine whether p22phox and nox-1 interact to form a functional oxidase in VSMCs. Our preliminary data indicate that both oxidase components are essential for 02- production, but we do not know if they function in concert. In the second specific aim, we will define the hypertrophy-related, redox-sensitive molecular targets of angiotensin 11-stimulated, NAD(P)H oxidase-derived ROS. We have previously shown that angiotensin II stimulation of the kinases c-Src, p38mitogen-activated protein kinase, and Akt are all mediated by ROS. We now plan to extend these observations to signaling pathways immediately upstream and downstream of Akt. Finally, we plan tc assess the role of redox-sensitive, growth-related signaling pathways in transgenic animal models of altered vascular oxidative stress. For these experiments, we will take advantage of new transgenic lines being developed at Emory in which p22phox, nox-1 or catalase are over expressed in vascular smooth muscle. These studies will provide insight into the mechanisms by which ROS mediate vascular function. The identification of cellular events involved in controlling the oxidative environment of VSMC may ultimately lead to the development of specific therapeutic interventions for vascular diseases characterized by abnormal vascular smooth muscle growth.